Phylogenetically diverse Bradyrhizobium genospecies nodulate Bambara groundnut (Vigna subterranea L. Verdc) and soybean (Glycine max L. Merril) in the northern savanna zones of Ghana.

A total of 102 bacterial strains isolated from nodules of three Bambara groundnut and one soybean cultivars grown in nineteen soil samples collected from northern Ghana were characterized using multilocus gene sequence analysis. Based on a concatenated sequence analysis (glnII-rpoB-recA-gyrB-atpD-dnaK), 54 representative strains were distributed in 12 distinct lineages, many of which were placed mainly in the Bradyrhizobium japonicum and Bradyrhizobium elkanii supergroups. Twenty-four of the 54 representative strains belonged to seven putative novel species, while 30 were conspecific with four recognized Bradyrhizobium species. The nodA phylogeny placed all the representative strains in the cosmopolitan nodA clade III. The strains were further separated in seven nodA subclusters with reference strains mainly of African origin. The nifH phylogeny was somewhat congruent with the nodA phylogeny, but both symbiotic genes were mostly incongruent with the core housekeeping gene phylogeny indicating that the strains acquired their symbiotic genes horizontally from distantly related Bradyrhizobium species. Using redundancy analysis, the distribution of genospecies was found to be influenced by the edaphic factors of the respective sampling sites. In general, these results mainly underscore the high genetic diversity of Bambara groundnut-nodulating bradyrhizobia in Ghanaian soils and suggest a possible vast resource of adapted inoculant strains.

Competition for electrons favours N2 O reduction in denitrifying Bradyrhizobium isolates.

Bradyrhizobia are common members of soil microbiomes and known as N2 -fixing symbionts of economically important legumes. Many are also denitrifiers, which can act as sinks or sources for N2 O. Inoculation with compatible rhizobia is often needed for optimal N2 -fixation, but the choice of inoculant may have consequences for N2 O emission. Here, we determined the phylogeny and denitrification capacity of Bradyrhizobium strains, most of them isolated from peanut-nodules. Analyses of genomes and denitrification end-points showed that all were denitrifiers, but only ~1/3 could reduce N2 O. The N2 O-reducing isolates had strong preference for N2 O- over NO3 - -reduction. Such preference was also observed in a study of other bradyrhizobia and tentatively ascribed to competition between the electron pathways to Nap (periplasmic NO3 - reductase) and Nos (N2 O reductase). Another possible explanation is lower abundance of Nap than Nos. Here, proteomics revealed that Nap was instead more abundant than Nos, supporting the hypothesis that the electron pathway to Nos outcompetes that to Nap. In contrast, Paracoccus denitrificans, which has membrane-bond NO3 - reductase (Nar), reduced N2 O and NO3 - simultaneously. We propose that the control at the metabolic level, favouring N2 O reduction over NO3 - reduction, applies also to other denitrifiers carrying Nos and Nap but lacking Nar.

Response of Soil Bacterial Community Diversity and Composition to Time, Fertilization, and Plant Species in a Sub-Boreal Climate.

Pastures are an important part of crop and food systems in cold climates. Understanding how fertilization and plant species affect soil bacterial community diversity and composition is the key for understanding the role of soil bacteria in sustainable agriculture. To study the response of soil bacteria to different fertilization and cropping managements, a 3-year (2013-2015) field study was established. In the split-plot design, fertilizer treatment (unfertilized control, organic fertilizer, and synthetic fertilizer) was the main plot factor, and plant treatment [clear fallow, red clover (Trifolium pratense), timothy (Phleum pratense), and a mixture of red clover and timothy] was the sub-plot factor. Soil bacterial community diversity and composition, soil properties, and crop growth were investigated through two growing seasons in 2014 and 2015, with different nitrogen input levels. The community diversity measures (richness, Shannon diversity, and Shannon evenness) and composition changed over time (P < 0.05) and at different time scales. The community diversity was lower in 2014 than in 2015. The temporal differences were greater than the differences between treatments. The overall correlations of Shannon diversity to soil pH, NO 3 - , NH 4 + , and surplus nitrogen were positive and that of bacterial richness to crop dry matter yield was negative (P < 0.05). The major differences in diversity and community composition were found between fallow and planted treatments and between organic and synthetic fertilizer treatments. The differences between the planted plots were restricted to individual operational taxonomic units (OTUs). Soil moisture, total carbon content, and total nitrogen content correlated consistently with the community composition (P < 0.05). Compared to the unfertilized control, the nitrogen fertilizer loading enhanced the temporal change of community composition in pure timothy and in the mixture more than that in red clover, which further emphasizes the complexity of interactions between fertilization and cropping treatments on soil bacteria.

Genetically diverse lentil- and faba bean-nodulating rhizobia are present in soils across Central and Southern Ethiopia.

In total 196 bacterial isolates were obtained from root nodules of lentil (Lens culinaris) and faba bean (Vicia faba) grown on soil samples collected from 10 different sites in central and southern parts of Ethiopia. All isolates were identified as members of the genus Rhizobium by using recA gene sequence analysis. In the recA phylogenetic tree 195 rhizobial strains were classified into nine genospecies. The phylogeny of symbiotic genes nodC and nifH revealed five and six distinct groups respectively, largely dominated by symbiovar viciae. A multivariate analysis showed that environmental variables of the sampling sites considered in this study had more effect on the distribution and composition of the genospecies than the host legumes of the strains. Twenty representative strains, selected based on their isolation site, host plant and nodC group, were able to nodulate all lentil, faba bean, field pea (Pisum abyssinicum) and grass pea (Lathyrus sativus) plants in a greenhouse test in axenic conditions. The majority of the rhizobial strains were effective nitrogen-fixing symbionts for all tested legumes, indicating their potential to serve as broad host-range inoculants in agriculture. The present work suggests the presence of taxonomically and symbiotically diverse rhizobial species for legumes in the Viciae tribe in Ethiopia.

Effectiveness of nitrogen fixation in rhizobia.

Biological nitrogen fixation in rhizobia occurs primarily in root or stem nodules and is induced by the bacteria present in legume plants. This symbiotic process has fascinated researchers for over a century, and the positive effects of legumes on soils and their food and feed value have been recognized for thousands of years. Symbiotic nitrogen fixation uses solar energy to reduce the inert N2 gas to ammonia at normal temperature and pressure, and is thus today, especially, important for sustainable food production. Increased productivity through improved effectiveness of the process is seen as a major research and development goal. The interaction between rhizobia and their legume hosts has thus been dissected at agronomic, plant physiological, microbiological and molecular levels to produce ample information about processes involved, but identification of major bottlenecks regarding efficiency of nitrogen fixation has proven to be complex. We review processes and results that contributed to the current understanding of this fascinating system, with focus on effectiveness of nitrogen fixation in rhizobia.

Minimal standards for the description of new genera and species of rhizobia and agrobacteria.

Herein the members of the Subcommittee on Taxonomy of Rhizobia and Agrobacteria of the International Committee on Systematics of Prokaryotes review recent developments in rhizobial and agrobacterial taxonomy and propose updated minimal standards for the description of new species (and genera) in these groups. The essential requirements (minimal standards) for description of a new species are (1) a genome sequence of at least the proposed type strain and (2) evidence for differentiation from other species based on genome sequence comparisons. It is also recommended that (3) genetic variation within the species is documented with sequence data from several clearly different strains and (4) phenotypic features are described, and their variation documented with data from a relevant set of representative strains. Furthermore, it is encouraged that information is provided on (5) nodulation or pathogenicity phenotypes, as appropriate, with relevant gene sequences. These guidelines supplement the current rules of general bacterial taxonomy, which require (6) a name that conforms to the International Code of Nomenclature of Prokaryotes, (7) validation of the name by publication either directly in the International Journal of Systematic and Evolutionary Microbiology or in a validation list when published elsewhere, and (8) deposition of the type strain in two international culture collections in separate countries.

Bacterial community changes in response to oil contamination and perennial crop cultivation.

We investigated bacterial community dynamics in response to used motor oil contamination and perennial crop cultivation by 16S rRNA gene amplicon sequencing in a 4-year field study. Actinobacteria, Proteobacteria, Chloroflexi, Acidobacteria, and Gemmatimonadetes were the major bacterial phyla, and Rhodococcus was the most abundant genus. Initially, oil contamination decreased the overall bacterial diversity. Actinobacteria, Betaproteobacteria, and Gammaproteobacteria were sensitive to oil contamination, exhibiting clear succession with time. However, bacterial communities changed over time, regardless of oil contamination and crop cultivation. The abundance difference of most OTUs between oil-contaminated and non-contaminated plots remained the same in later sampling years after the initial abundance difference induced by oil spike. The abundances of three oil-favored actinobacteria (Lysinimonas, Microbacteriaceae, and Marmoricola) and one betaproteobacterium (Aquabacterium) changed in different manner over time in oil-contaminated and non-contaminated soil. We propose that these taxa are potential bio-indicators for monitoring recovery from motor oil contamination in boreal soil. The effect of crop cultivation on bacterial communities became significant only after the crops achieved stable growth, likely associated with plant material decomposition by Bacteroidetes, Armatimonadetes and Fibrobacteres.

Draft genome sequences of Bradyrhizobium shewense sp. nov. ERR11T and Bradyrhizobium yuanmingense CCBAU 10071T.

The type strain of the prospective 10.1601/nm.30737 sp. nov. ERR11T, was isolated from a nodule of the leguminous tree Erythrina brucei native to Ethiopia. The type strain 10.1601/nm.1463 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+10071 T, was isolated from the nodules of Lespedeza cuneata in Beijing, China. The genomes of ERR11T and 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+10071 T were sequenced by DOE-JGI and deposited at the DOE-JGI genome portal as well as at the European Nucleotide Archive. The genome of ERR11T is 9,163,226 bp in length and has 102 scaffolds, containing 8548 protein-coding and 86 RNA genes. The 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+10071 T genome is arranged in 108 scaffolds and consists of 8,201,522 bp long and 7776 protein-coding and 85 RNA genes. Both genomes contain symbiotic genes, which are homologous to the genes found in the complete genome sequence of 10.1601/nm.24498 10.1601/strainfinder?urlappend=%3Fid%3DUSDA+110 T. The genes encoding for nodulation and nitrogen fixation in ERR11T showed high sequence similarity with homologous genes found in the draft genome of peanut-nodulating 10.1601/nm.27386 10.1601/strainfinder?urlappend=%3Fid%3DLMG+26795 T. The nodulation genes nolYA-nodD2D1YABCSUIJ-nolO-nodZ of ERR11T and 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+10071 T are organized in a similar way to the homologous genes identified in the genomes of 10.1601/strainfinder?urlappend=%3Fid%3DUSDA+110 T, 10.1601/nm.25806 10.1601/strainfinder?urlappend=%3Fid%3DUSDA+4 and 10.1601/nm.1462 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+05525. The genomes harbor hupSLCFHK and hypBFDE genes that code the expression of hydrogenase, an enzyme that helps rhizobia to uptake hydrogen released by the N2-fixation process and genes encoding denitrification functions napEDABC and norCBQD for nitrate and nitric oxide reduction, respectively. The genome of ERR11T also contains nosRZDFYLX genes encoding nitrous oxide reductase. Based on multilocus sequence analysis of housekeeping genes, the novel species, which contains eight strains formed a unique group close to the 10.1601/nm.25806 branch. Genome Average Nucleotide Identity (ANI) calculated between the genome sequences of ERR11T and closely related sequences revealed that strains belonging to 10.1601/nm.25806 branch (10.1601/strainfinder?urlappend=%3Fid%3DUSDA+4 and 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+15615), were the closest strains to the strain ERR11T with 95.2% ANI. Type strain ERR11T showed the highest DDH predicted value with 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+15615 (58.5%), followed by 10.1601/strainfinder?urlappend=%3Fid%3DUSDA+4 (53.1%). Nevertheless, the ANI and DDH values obtained between ERR11T and 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+15615 or 10.1601/strainfinder?urlappend=%3Fid%3DUSDA+4 were below the cutoff values (ANI ≥ 96.5%; DDH ≥ 70%) for strains belonging to the same species, suggesting that ERR11T is a new species. Therefore, based on the phylogenetic analysis, ANI and DDH values, we formally propose the creation of 10.1601/nm.30737 sp. nov. with strain ERR11T (10.1601/strainfinder?urlappend=%3Fid%3DHAMBI+3532 T=10.1601/strainfinder?urlappend=%3Fid%3DLMG+30162 T) as the type strain.

Draft genome sequence of type strain HBR26T and description of Rhizobium aethiopicum sp. nov.

Rhizobium aethiopicum sp. nov. is a newly proposed species within the genus Rhizobium. This species includes six rhizobial strains; which were isolated from root nodules of the legume plant Phaseolus vulgaris growing in soils of Ethiopia. The species fixes nitrogen effectively in symbiosis with the host plant P. vulgaris, and is composed of aerobic, Gram-negative staining, rod-shaped bacteria. The genome of type strain HBR26T of R. aethiopicum sp. nov. was one of the rhizobial genomes sequenced as a part of the DOE JGI 2014 Genomic Encyclopedia project designed for soil and plant-associated and newly described type strains. The genome sequence is arranged in 62 scaffolds and consists of 6,557,588 bp length, with a 61% G + C content and 6221 protein-coding and 86 RNAs genes. The genome of HBR26T contains repABC genes (plasmid replication genes) homologous to the genes found in five different Rhizobium etli CFN42T plasmids, suggesting that HBR26T may have five additional replicons other than the chromosome. In the genome of HBR26T, the nodulation genes nodB, nodC, nodS, nodI, nodJ and nodD are located in the same module, and organized in a similar way as nod genes found in the genome of other known common bean-nodulating rhizobial species. nodA gene is found in a different scaffold, but it is also very similar to nodA genes of other bean-nodulating rhizobial strains. Though HBR26T is distinct on the phylogenetic tree and based on ANI analysis (the highest value 90.2% ANI with CFN42T) from other bean-nodulating species, these nod genes and most nitrogen-fixing genes found in the genome of HBR26T share high identity with the corresponding genes of known bean-nodulating rhizobial species (96-100% identity). This suggests that symbiotic genes might be shared between bean-nodulating rhizobia through horizontal gene transfer. R. aethiopicum sp. nov. was grouped into the genus Rhizobium but was distinct from all recognized species of that genus by phylogenetic analyses of combined sequences of the housekeeping genes recA and glnII. The closest reference type strains for HBR26T were R. etli CFN42T (94% similarity of the combined recA and glnII sequences) and Rhizobium bangladeshense BLR175T (93%). Genomic ANI calculation based on protein-coding genes also revealed that the closest reference strains were R. bangladeshense BLR175T and R. etli CFN42T with ANI values 91.8 and 90.2%, respectively. Nevertheless, the ANI values between HBR26T and BLR175T or CFN42T are far lower than the cutoff value of ANI (> = 96%) between strains in the same species, confirming that HBR26T belongs to a novel species. Thus, on the basis of phylogenetic, comparative genomic analyses and ANI results, we formally propose the creation of R. aethiopicum sp. nov. with strain HBR26T (=HAMBI 3550T=LMG 29711T) as the type strain. The genome assembly and annotation data is deposited in the DOE JGI portal and also available at European Nucleotide Archive under accession numbers FMAJ01000001-FMAJ01000062.

Diverse Mesorhizobium bacteria nodulate native Astragalus and Oxytropis in arctic and subarctic areas in Eurasia.

Rhizobia nodulating native Astragalus and Oxytropis spp. in Northern Europe are not well-studied. In this study, we isolated bacteria from nodules of four Astragalus spp. and two Oxytropis spp. from the arctic and subarctic regions of Sweden and Russia. The phylogenetic analyses were performed by using sequences of three housekeeping genes (16S rRNA, rpoB and recA) and two accessory genes (nodC and nifH). The results of our multilocus sequence analysis (MLSA) of the three housekeeping genes tree showed that all the 13 isolates belonged to the genus Mesorhizobium and were positioned in six clades. Our concatenated housekeeping gene tree also suggested that the isolates nodulating Astragalus inopinatus, Astragalus frigidus, Astragalus alpinus ssp. alpinus and Oxytropis revoluta might be designated as four new Mesorhizobium species. The 13 isolates were grouped in three clades in the nodC and nifH trees. 15N analysis suggested that the legumes in association with these isolates were actively fixing nitrogen.

Microbial cooperation in the rhizosphere improves liquorice growth under salt stress.

Liquorice (Glycyrrhiza uralensis Fisch.) is one of the most widely used plants in food production, and it can also be used as an herbal medicine or for reclamation of salt-affected soils. Under salt stress, inhibition of plant growth, nutrient acquisition and symbiotic interactions between the medicinal legume liquorice and rhizobia have been observed. We recently evaluated the interactions between rhizobia and root-colonizing Pseudomonas in liquorice grown in potting soil and observed increased plant biomass, nodule numbers and nitrogen content after combined inoculation compared to plants inoculated with Mesorhizobium alone. Several beneficial effects of microbes on plants have been reported; studies examining the interactions between symbiotic bacteria and root-colonizing Pseudomonas strains under natural saline soil conditions are important, especially in areas where a hindrance of nutrients and niches in the rhizosphere are high. Here, we summarize our recent observations regarding the combined application of rhizobia and Pseudomonas on the growth and nutrient uptake of liquorice as well as the salt stress tolerance mechanisms of liquorice by a mutualistic interaction with microbes. Our observations indicate that microbes living in the rhizosphere of liquorice can form a mutualistic association and coordinate their involvement in plant adaptations to stress tolerance. These results support the development of combined inoculants for improving plant growth and the symbiotic performance of legumes under hostile conditions.

Evolution and taxonomy of native mesorhizobia nodulating medicinal Glycyrrhiza species in China.

Previously, 159 bacterial strains were isolated from the root nodules of wild perennial Glycyrrhiza legume species grown on 40 sites in central and north-western China, in which 57 strains were classified as "true symbionts" belonging to the genus Mesorhizobium based on amplified fragment length polymorphism (AFLP) genomic fingerprinting and partial sequences of the 16S rRNA gene [20]. In the present work, the phylogeny of Glycyrrhiza nodulating mesorhizobia was further examined by multilocus sequence analysis (MLSA). The concatenated gene tree of three housekeeping genes (16S rRNA, recA, and rpoB) of 59 strains including the 29 mesorhizobial test strains and 30 type mesorhizobial species, was constructed applying the maximum likelihood method and Bayesian inference. In the concatenated gene tree, the 29 test strains were distributed in seven separate clades. Seventeen test strains clustered with Mesorhizobium tianshanense, Mesorhizobium temperatum, Mesorhizobium muleiense, and Mesorhizobium alhagi with high bootstrap support (BS>85%). Eight test strains did not cluster with any of the described Mesorhizobium species. Based on the results, we proposed these eight test strains might belong to a putative new species of the genus Mesorhizobium. The sequences of three accessory genes (nodA, nodC, and nifH) of the test strains were also analyzed and were compared with those of representatives of the 30 described mesorhizobial species. The results showed that mesorhizobia involved in symbiosis with Glycyrrhiza plants probably have acquired some genetic material from other rhizobia in co-evolution with Glycyrrhiza and other legume species.

Divergent genes in potential inoculant Sinorhizobium strains are related to DNA replication, recombination, and repair.

To serve as inoculants of legumes, nitrogen-fixing rhizobium strains should be competitive and tolerant of diverse environments. We hybridized the genomes of symbiotically efficient and salt tolerant Sinorhizobium inoculant strains onto the Sinorhizobium meliloti Rm1021 microarray. The number of variable genes, that is, divergent or putatively multiplied genes, ranged from 503 to 1556 for S. meliloti AK23, S. meliloti STM 1064 and S. arboris HAMBI 1552. The numbers of divergent genes affiliated with the symbiosis plasmid pSymA and related to DNA replication, recombination and repair were significantly higher than expected. The variation was mainly in the accessory genome, implying that it was important in shaping the adaptability of the strains.

A synergistic interaction between salt-tolerant Pseudomonas and Mesorhizobium strains improves growth and symbiotic performance of liquorice (Glycyrrhiza uralensis Fish.) under salt stress.

Chinese liquorice (Glycyrrhiza uralensis Fish.) is a salt-tolerant medicinal legume that could be utilized for bioremediation of salt-affected soils. We studied whether co-inoculation of the symbiotic Mesorhizobium sp. strain NWXJ19 or NWXJ31 with the plant growth-promoting Pseudomonas extremorientalis TSAU20 could restore growth, nodulation, and shoot/root nitrogen contents of salt-stressed G. uralensis, which was grown in potting soil and irrigated with 0, 50, and 75 mM NaCl solutions under greenhouse conditions. Irrigation with NaCl solutions clearly retarded the growth of uninoculated liquorice, and the higher the NaCl concentration (75 and 100 mM NaCl), the more adverse is the effect. The two Mesorhizobium strains, added either alone or in combination with P. extremorientalis TSAU20, responded differently to the salt levels used. The strain NWXJ19 was a good symbiont for plants irrigated with 50 mM NaCl, whereas the strain NWXJ31 was more efficient for plants irrigated with water or 75 mM NaCl solution. P. extremorientalis TSAU20 combined with single Mesorhizobium strains alleviated the salt stress of liquorice plants and improved yield and nodule numbers significantly in comparison with single-strain-inoculated liquorice. Both salt stress and inoculation raised the nitrogen content of shoots and roots. The nitrogen contents were at their highest, i.e., 30 and 35 % greater compared to non-stressed uninoculated plants, when plants were inoculated with P. extremorientalis TSAU20 and Mesorhizobium sp. NWXJ31 as well as irrigated with 75 mM NaCl solution. From this study, we conclude that dual inoculation with plant growth-promoting rhizobacteria could be a new approach to improve the tolerance of G. uralensis to salt stress, thereby improving its suitability for the remediation of saline lands.

Characterization of successional changes in bacterial community composition during bioremediation of used motor oil-contaminated soil in a boreal climate.

The widespread use of motor oil makes it a notable risk factor to cause scattered contamination in soil. The monitoring of microbial community dynamics can serve as a comprehensive tool to assess the ecological impact of contaminants and their disappearance in the ecosystem. Hence, a field study was conducted to monitor the ecological impact of used motor oil under different perennial cropping systems (fodder galega, brome grass, galega-brome grass mixture and bare fallow) in a boreal climate zone. Length heterogeneity PCR characterized a successional pattern in bacterial community following oil contamination over a four-year bioremediation period. Soil pH and electrical conductivity were associated with the shifts in bacterial community composition. Crops had no detectable effect on bacterial community composition or complexity. However, the legume fodder galega increased soil microbial biomass, expressed as soil total DNA. Oil contamination induced an abrupt change in bacterial community composition at the early stage, yet the effect did not last as long as the oil in soil. The successional variation in bacterial community composition can serve as a sensitive ecological indicator of oil contamination and remediation in situ.

Perennial crop growth in oil-contaminated soil in a boreal climate.

Soil contamination by petroleum hydrocarbons is a global problem. Phytoremediation by plants and their associated microorganisms is a cost-effective strategy to degrade soil contaminants. In boreal regions the cool climate limits the efficiency of phytoremediation. The planting of oil-tolerant perennial crops, especially legumes, in oil-contaminated soil holds promise for great economic benefits for bioenergy and bio-fertilizer production while accelerating the oil degradation process. We established a multi-year field experiment to study the ecological and agronomic feasibility of phytoremediation by a legume (fodder galega) and a grass (smooth brome) in a boreal climate. In 40 months, soil oil content decreased by 73%-92%, depending on the crop type. The oil degradation followed first-order kinetics with the reduction rates decreasing as follows: bare fallow > galega-brome grass mixture > brome grass > galega. Surprisingly, the presence of oil enhanced crop dry matter and nitrogen yield, particularly in the fourth year. The unfertilized galega-brome grass mixture out-yielded the N-fertilized pure grass swards over years by an average of 33%. Thus, a perennial legume-grass mixture is both ecologically and agronomically sustainable as a cropping system to alleviate soil contamination in the boreal zone, with considerable potential for bioenergy and bio-fertilizer production.

Genomic features separating ten strains of Neorhizobium galegae with different symbiotic phenotypes.

BACKGROUND: The symbiotic phenotype of Neorhizobium galegae, with strains specifically fixing nitrogen with either Galega orientalis or G. officinalis, has made it a target in research on determinants of host specificity in nitrogen fixation. The genomic differences between representative strains of the two symbiovars are, however, relatively small. This introduced a need for a dataset representing a larger bacterial population in order to make better conclusions on characteristics typical for a subset of the species. In this study, we produced draft genomes of eight strains of N. galegae having different symbiotic phenotypes, both with regard to host specificity and nitrogen fixation efficiency. These genomes were analysed together with the previously published complete genomes of N. galegae strains HAMBI 540T and HAMBI 1141. RESULTS: The results showed that the presence of an additional rpoN sigma factor gene in the symbiosis gene region is a characteristic specific to symbiovar orientalis, required for nitrogen fixation. Also the nifQ gene was shown to be crucial for functional symbiosis in both symbiovars. Genome-wide analyses identified additional genes characteristic of strains of the same symbiovar and of strains having similar plant growth promoting properties on Galega orientalis. Many of these genes are involved in transcriptional regulation or in metabolic functions. CONCLUSIONS: The results of this study confirm that the only symbiosis-related gene that is present in one symbiovar of N. galegae but not in the other is an rpoN gene. The specific function of this gene remains to be determined, however. New genes that were identified as specific for strains of one symbiovar may be involved in determining host specificity, while others are defined as potential determinant genes for differences in efficiency of nitrogen fixation.

Revised phylogeny of Rhizobiaceae: proposal of the delineation of Pararhizobium gen. nov., and 13 new species combinations.

The family Rhizobiaceae accommodates the seven genera Rhizobium, Neorhizobium, Allorhizobium, Agrobacterium, Ensifer (syn. Sinorhizobium), Shinella and Ciceribacter. However, several so-called Rhizobium species do not exhibit robust phylogenetic positions. Rhizobium is extremely heterogeneous and is in need of major revision. Therefore, a phylogenetic examination of the family Rhizobiaceae by multilocus sequence analysis (MLSA) of four housekeeping genes among 100 strains of the family was undertaken. Based on the results we propose the delineation of the new genus Pararhizobium in the Rhizobiaceae family, and 13 new species combinations: Agrobacterium nepotum comb. nov., Agrobacterium pusense comb. nov., Agrobacterium skierniewicense comb. nov., Allorhizobium vitis comb. nov., Allorhizobium taibaishanense comb. nov., Allorhizobium paknamense comb. nov., Allorhizobium oryzae comb. nov., Allorhizobium pseudoryzae comb. nov., Allorhizobium borbori comb. nov., Pararhizobium giardinii comb. nov., Pararhizobium capsulatum comb. nov., Pararhizobium herbae comb. nov., and Pararhizobium sphaerophysae comb. nov.

Genome sequencing of two Neorhizobium galegae strains reveals a noeT gene responsible for the unusual acetylation of the nodulation factors.

BACKGROUND: The species Neorhizobium galegae comprises two symbiovars that induce nodules on Galega plants. Strains of both symbiovars, orientalis and officinalis, induce nodules on the same plant species, but fix nitrogen only in their own host species. The mechanism behind this strict host specificity is not yet known. In this study, genome sequences of representatives of the two symbiovars were produced, providing new material for studying properties of N. galegae, with a special interest in genomic differences that may play a role in host specificity. RESULTS: The genome sequences confirmed that the two representative strains are much alike at a whole-genome level. Analysis of orthologous genes showed that N. galegae has a higher number of orthologs shared with Rhizobium than with Agrobacterium. The symbiosis plasmid of strain HAMBI 1141 was shown to transfer by conjugation under optimal conditions. In addition, both sequenced strains have an acetyltransferase gene which was shown to modify the Nod factor on the residue adjacent to the non-reducing-terminal residue. The working hypothesis that this gene is of major importance in directing host specificity of N. galegae could not, however, be confirmed. CONCLUSIONS: Strains of N. galegae have many genes differentiating them from strains of Agrobacterium, Rhizobium and Sinorhizobium. However, the mechanism behind their ecological difference is not evident. Although the final determinant for the strict host specificity of N. galegae remains to be identified, the gene responsible for the species-specific acetylation of the Nod factors was identified in this study. We propose the name noeT for this gene to reflect its role in symbiosis.

Soil mesocosm studies on atrazine bioremediation.

Accumulation of pesticides in the environment causes serious issues of contamination and toxicity. Bioremediation is an ecologically sound method to manage soil pollution, but the bottleneck here, is the successful scale-up of lab-scale experiments to field applications. This study demonstrates pilot-scale bioremediation in tropical soil using atrazine as model pollutant. Mimicking field conditions, three different bioremediation strategies for atrazine degradation were explored. 100 kg soil mesocosms were set-up, with or without atrazine application history. Natural attenuation and enhanced bioremediation were tested, where augmentation with an atrazine degrading consortium demonstrated best pollutant removal. 90% atrazine degradation was observed in six days in soil previously exposed to atrazine, while soil without history of atrazine use, needed 15 days to remove the same amount of amended atrazine. The bacterial consortium comprised of 3 novel bacterial strains with different genetic atrazine degrading potential. The progress of bioremediation was monitored by measuring the levels of atrazine and its intermediate, cyanuric acid. Genes from the atrazine degradation pathway, namely, atzA, atzB, atzD, trzN and trzD were quantified in all mesocosms for 60 days. The highest abundance of all target genes was observed on the 6th day of treatment. trzD was observed in the bioaugmented mesocosms only. The bacterial community profile in all mesocosms was monitored by LH-PCR over a period of two months. Results indicate that the communities changed rapidly after inoculation, but there was no drastic change in microbial community profile after 1 month. Results indicated that efficient bioremediation of atrazine using a microbial consortium could be successfully up-scaled to pilot scale.